Microvascular hyperpermeability is a hallmark of the systemic inflammatory response syndrome (SIRS) present in 93% of intensive care patients. SIRS severely complicates resuscitation of trauma victims and worsens clinical outcomes, frequently progressing to multiple organ failure. The estimated annual US health care cost burden associated with SIRS is $16.7 billion. Despite advances in understanding of inflammation- induced microvascular hyperpermeability, the mechanisms that restore microvascular permeability to normal following an inflammatory challenge are unknown. Moreover, existing clinical treatments are not effective for restoration of microvascular barrier integrity once the inflammatory cascade has been initiated. We propose a novel paradigm of signaling mechanisms responsible for restoration of microvascular integrity following inflammation caused by traumatic injury. Our preliminary data show that administration of a polyamine-coated, cell permeable form of Rnd3, a Rho family small GTPase, reduces microvascular hyperpermeability in a rat model of hemorrhagic shock. We also have observed with live cell imaging of endothelial cells that lamellipodia formation and turnover represent a previously uncharacterized cell behavior important for normal endothelial barrier integrity. We hypothesize that Rnd3 promotes endothelial barrier restoration after inflammatory challenges by inhibiting RhoA/ROCK-mediated cell contraction and activating Rac1-mediated enhancement of intercellular junction integrity. Our specific aims are to: 1) Test the prediction that Ser phosphorylation and membrane targeting of Rnd3 are required for Rnd3 to reduce microvascular hyperpermeability;2) To test the prediction that Rnd3 promotes negative feedback inhibition of ROCK-mediated cell contraction, which enhances endothelial barrier integrity;3) To test the prediction that Rnd3 enhances Rac1-mediated lamellipodia formation and stabilization of VE-cadherin at junctions, promoting endothelial barrier integrity. The proposed novel, integrated approach capitalizes on a refined and relevant rat model of hemorrhagic shock combined with intravital microscopy of the in vivo mesenteric microcirculation and isolated venule methods to assess microvascular permeability. We will explore the central role of Rnd3 by employing an innovative method to deliver cell permeable Rnd3 protein to the mesenteric microcirculation. Cultured endothelial cell monolayer permeability models, imaging of GFP-actin and GFP-VE-cadherin dynamics in live endothelial cells, and biochemical studies will support the in vivo and isolated venule studies. The results of this study will enable us to develop a new theory of how endothelial barrier function can be restored during inflammation, which will serve as the foundation for novel therapies. Discovery of targets that can be used to resolve microvascular hyperpermeability will revolutionize the treatment of trauma patients, and will also create new opportunities to treat edema associated with a wide range of diseases. PUBLIC HEALTH RELEVANCE: Systemic inflammation caused by injury results in microvascular leakage that worsens outcome in trauma patients. Understanding the cellular and molecular mechanisms that lead to resolution of microvascular leakage is necessary to develop new therapies to ameliorate complications from systemic inflammation.